The normal human complement of chromosomes consists of the sex chromosomes (designated X and Y) and 22 autosomes (numbered 1-22). It has been estimated that a minimum of 1 in 10 human conceptions has a chromosome abnormality. As a general rule, an abnormal number of sex chromosomes is not lethal, although infertility can result. In contrast, an abnormal number of autosomes typically results in early death. Of the three autosomal trisomies found in live-born babies (trisomy 21, 18 and 13), only individuals with trisomy 21 (more commonly known as Down syndrome), survive past infancy.
Although Down syndrome is easily diagnosed after birth, prenatal diagnosis is problematic. To date, karyotyping of fetal cells is the only definitive method for diagnosing the disorder. Typically, fetal cells for karyotyping are isolated by invasive procedures such as amniocentesis or chorionic villus sampling. Amniocentesis and chorionic villus sampling are performed at about 15-18 weeks and 9-12 weeks gestation, respectively. A fetal loss rate of about 0.2-0.5% is associated with amniocentesis; Chorionic villus sampling reportedly has a fetal loss rate of between about 1-2%.
The finite risks of miscarriage of a normal fetus, as well as the high costs associated with the above-described procedures, have limited the widespread application of amniocentesis and chorionic villus sampling for the routine screening of pregnant women. Thus, diagnosis of Down syndrome during pregnancy has been limited to women identified as having a high risk for carrying a Down syndrome-affected child. In practice, this has meant limiting the tests for Down syndrome to women who are age 35 and older or who have a history of pregnancies associated with chromosomal damage. Approximately 20% of Down Syndrome children are born to women 35 years old or older.
In view of the relatively high incidence of Down syndrome infants born to women age 35 and under, efforts have been made to develop screening procedures to identify women having an elevated risk of carrying a Down syndrome-affected child. These methods include sonography and/or the measurement in maternal blood of multiple markers indicative of a Down syndrome pregnancy.
The association of various maternal serum markers with Down syndrome has been reported. In general, the absolute concentration of a marker is not determined. Rather, the amount of marker is expressed as a multiple of a normal (i.e., non-Down syndrome pregnancy) median ("MoMs") at a known gestation time. Thus, the average MoM value for a marker indicates the extent of marker increase or decrease for a Down syndrome pregnancy compared to a normal pregnancy.
A comparison of the ability of twelve markers to distinguish a Down syndrome pregnancy from a normal pregnancy has been reported (Cuckle, H., Clin. Chem. 38(9):1687 1689 (1992). The ability of different markers to distinguish a Down syndrome pregnancy was based upon a determination of the number of standard deviations from which each marker MoM valve for a Down syndrome pregnancy differed from the MoM value obtained for a normal pregnancy. The most discriminating markers reportedly were neutrophil alkaline phosphatase, chorionic gonadotropin (hCG) and free-beta-hCG, with alpha-fetoprotein (AFP), .mu.Ec (unconjugated estriol) and pregnancy-specific beta.sub.1 -glycoprotein. However, the author further concluded that in practice, hCG (intact, total, or free-beta) was the marker of first choice because the cytochemical assay of neutrophil alkaline phosphatase is not suitable for large throughput of samples.
The term "cytochemical assay" refers to a method for studying the presence and/or distribution of intracellular components. In general, this is accomplished by contacting a plurality of fixed cells with a stain or other reagent to yield a visually-detectable product. In the cytochemical assay of neutrophil alkaline phosphatase, a blood sample smear is fixed, lysed and bound with a substrate of the enzyme to give an insoluble product which appears as colored granules in the cytoplasm of neutrophils. Each step of the assay is labor-intensive and involves a high degree of subjectivity. Thus, for example, a laboratory technologist must manually prepare and stain the blood sample smear, visually select neutrophils from other cells in the optical field, assess the amount of color present in the stained cells and compare this visually-perceived amount of color to the color of control blood smears having amounts of neutrophil alkaline phosphatase indicative of the presence or absence of a Down syndrome pregnancy. It will be readily apparent that the above-described procedure is quite time-consuming. Indeed, it may take 10-15 minutes to perform this assay on one cell sample from one individual.
Although cytochemical assays for the classification of blood cells have been automated to some extent (see e.g., Parthenis, K. and Metazaki-Kossionides, C., J. Biomed. Eng. 14:287-292 (1992) and Pooh, S., et al., Cytometry 13:766-774 (1992)), automated cytochemical analysis has not been applied to the quantitation of intracellular analytes for the identification of pregnant women having an elevated likelihood of carrying a Down syndrome-affected fetus.
However, automated cytochemical analysis has not been applied to the quantitation of other intracellular analytes, because of the difficulty encountered in isolating the particular cells or cell structures of interest within a cell sample. Consider, for example, the assay of neutrophil alkaline phosphatase discussed above. The optical field viewed by the Technician includes red blood cells, debris and other objects, as well as the sought-after neutrophils. Furthermore, denser portions of a smear can include overlapped or clumped objects that appear to be single objects.
Therefore, it is a general goal of the present invention to provide a method and apparatus for performing cytochemical analysis in order to quantify the level of an intracellular analyte within selected cell types which can be found within a cell sample.
A more particular goal of the present invention is to provide a method and apparatus suitable for performing automated cytochemical analysis for quantifying the level of intracellular analytes suitable for identifying preleukemic states and pregnant women having an elevated likelihood of carrying a Down Syndrome-affected fetus.
Another goal is to provide a method of automatically isolating one image object within an optical field containing a variety of types of image objects, some of which are of interest.